异丙酚联合缺血预处理对大鼠肺缺血再灌注损伤时细胞凋亡的影响

目的 探讨异丙酚联合缺血预处理对大鼠肺缺血苒灌注损伤时细胞凋亡的影响.方法 雄性SD大鼠50只,200～250 g,随机分为5组(n=10):假手术组(S组)、缺血再灌注组(IR组)、异丙酚组(P组)、缺血预处理组(IP组)和异丙酚+缺血预处理组(P+IP组).阻断右肺门1 h后再灌注2 h制备大鼠单肺原位热缺血再灌注模型,P组夹闭右肺门前30 min持续静脉输注异丙酚30 mg·kg-1·h-1;IP组夹闭右肺门前先进行夹闭5 min,再灌注5 min,反复3次的缺血预处理;P+IP组夹闭肺门前30 min静脉输注异丙酚30 mg·kg-1·h-1和缺血预处理.于再灌注2 h时处死大鼠,取右肺下叶肺组织,光镜下观察肺组织病理学结果 ,计算肺损伤定量评价指标(IQA);检测肺凋亡细胞,计算肺细胞凋亡指数(AI);采用免疫组化法检测Bcl-2、Bax蛋白表达;IQA、Bcl-2/Bax蛋白比值与AI作直线相关分析.结果 与S组比较,IR组、P组、IP组和P+IP组再灌注后IQA、AI均明显升高,IR组Bcl-2、Bax蛋白表达上调,Bcl-2/Bax蛋白比值降低(P<0.01);与IR组比较,P组、IP组和P+IP组IQA、AI均明显降低,Bcl-2蛋白表达水平、Bcl-2/Bax蛋白比值升高,IP组、P+IP组Bax蛋白表达下调(P<0.01),P组差异无统计学意义(P>0.05);与P组和IP组比较,P+IP组IQA及AI降低,Bcl-2/Bax蛋白比值升高(P<0.05);IQA与AI呈正相关(r=0.951,P<0.01);AI与Bcl-2/Bax蛋白比值呈负相关(r=-0.851,P<0.01).结论 异丙酚联合缺血预处理可通过调节Bcl-2和Bax蛋白的表达抑制细胞凋亡,减轻肺缺血再灌注损伤.     

异丙酚对大鼠小肠缺血再灌注时粘膜上皮细胞凋亡的影响

目的 探讨异丙酚对大鼠小肠缺血再灌注时粘膜上皮细胞凋亡的影响及其机制.方法 健康Wistar大鼠24只,随机分为3组(n=8):假手术组(S组)、缺血再灌注组(I/R组)和异丙酚+缺血再灌注组(P+I/R组).夹闭肠系膜上动脉,缺血1 h,再灌注2 h,制备小肠缺血再灌注损伤模型.S组和I/R组缺血前10 min开始经股静脉持续输注生理盐水10 ml·kg-1·h-1,P+I/R组静脉注射异丙酚10 mg/kg,以后持续输注异丙酚10 mg·kg-1·h-1.取空肠组织3 cm,常规制备全层石蜡切片,行HE染色,光镜下观察小肠组织病理学;免疫组化法检测Bcl-2、Bax蛋白的表达;TUNEL法检测小肠粘膜上皮细胞凋亡,计数凋亡细胞及总细胞,计算小肠粘膜上皮细胞凋亡指数.结果 与S组比较,I/R组和P+I/R组Bcl-2和Bax蛋白表达上调,Bcl-2/Bax增加,小肠组织损伤程度增强,小肠粘膜上皮细胞凋亡指数增高(P＜0.01或0.05);与I/R组比较,P+I/R组Bcl-2蛋白表达上调,Bax蛋白表达下调,小肠组织损伤程度减轻,小肠粘膜上皮细胞凋亡指数降低(P＜0.01).结论 异丙酚可减轻大鼠小肠缺血再灌注损伤时粘膜上皮细胞凋亡,其机制与上调Bcl-2基因的表达和下调Bax基因的表达有关.     

异丙酚对失血性休克/复苏兔胃黏膜细胞凋亡的影响

目的 评价异丙酚对失血性休克/复苏兔胃黏膜细胞凋亡的影响.方法 健康成年雄性新西兰大白兔100只,体重2.5～3.0kg,采用随机数字表法,将其随机分为5组(n=20):假手术组(S组)、模型组(M组)及异丙酚不同给药时机组P1组:预先给药组、P2组:后处理组和P3组:治疗组.采用股动脉放血及股静脉回输血、输液法制备失血性休克/复苏诱发胃黏膜损伤模型,MAP降至35～40 mm Hg并维持60 min.P1组、P2组和P3组分别于放血前10 min、复苏开始前10 min及复苏开始后20 min时静脉注射异丙酚5mG/kg后,以20mg·kg-1·h-1静脉输注至复苏开始后90min,S组和M组给予等容量生理盐水.复苏开始后90 min时取胃组织,肉眼下观察胃黏膜损伤情况,采用TUNEL法和免疫组化法分别测定胃黏膜细胞凋亡及Bcl-2和Bax蛋白表达.结果 与S组比较,余4组胃黏膜有不同程度损伤,凋亡指数(AI)及Bax蛋白表达升高,而Bcl-2蛋白表达及Bcl-2/Bax比值降低(P＜0.05或0.01).与M组比较,P1组、P2组和P3组胃黏膜损伤减轻,AI降低,P1组和P2组Bax蛋白表达降低,而Bcl-2蛋白表达和Bcl-2/Bax比值升高(P＜0.05或0.01).与P3组比较,P1组胃黏膜损伤减轻,AI和Bax蛋白表达降低,而Bcl-2蛋白表达和Bcl-2/Bax比值升高(P＜0.05).结论 异丙酚预先给药和后处理通过上调Bcl-2蛋白表达和下调Bax蛋白表达减轻失血性休克/复苏兔胃黏膜细胞凋亡.     

异丙酚对急性肺栓塞大鼠肺细胞凋亡的影响

Objective To evaluate the effect of propofol on lung cell apoptosis induced by acute pulmonary thromboembolism (APTE) .Methods Forty male SD rats weighing 280-300 g were randomly divided into 5 groups ( n = 8 each) : group Ⅰ sham operation ( group S) ; group ⅡAPTE and 3 propofol groups ( group P1-3). APTE was produced by iv injection of auto-blood clots. Venous blood 0.2 ml was obtained from rat tail vein and placed in a sterile test tube which was kept in water bath at 37 ℃ overnight. The blood clot was cut into thrombi ( diameter 1 mm, length 5 mm) the next day. Fifteen thrombi in 2 ml of normal saline were injected into immediately after iv injection of auto-bloed clots. The animals were killed at the end of 4 h propofol infusion and lung specimens were obtained for determination of lung cell apoptosis rate by flow eytometry and expression of caspase-3, Bax, Bcl-2, Fas, FasL mRNA and protein by RT-PCR and Western blot.The expression of Bcl-2/Bax mRNA and protein was calculated. Results Compared with group S,APTE significantly increased the lung cell apoptosis rate, and expression of caspase-3, Bax, Fas, FasL and decreased the expression of Bcl-2 and Bcl-2/Bax. Propofol infusion significantly attenuated these APTE-induced changes. Conclusion Propofol can inhibit APTE-induced lung cell apoptosis by down-regulating the caspase-3, Fas and FasL expression and regulating the balance between Bcl-2 and Bax expression.     

异丙酚对急性肺栓塞大鼠肺细胞凋亡的影响

Objective To evaluate the effect of propofol on lung cell apoptosis induced by acute pulmonary thromboembolism (APTE) .Methods Forty male SD rats weighing 280-300 g were randomly divided into 5 groups ( n = 8 each) : group Ⅰ sham operation ( group S) ; group ⅡAPTE and 3 propofol groups ( group P1-3). APTE was produced by iv injection of auto-blood clots. Venous blood 0.2 ml was obtained from rat tail vein and placed in a sterile test tube which was kept in water bath at 37 ℃ overnight. The blood clot was cut into thrombi ( diameter 1 mm, length 5 mm) the next day. Fifteen thrombi in 2 ml of normal saline were injected into immediately after iv injection of auto-bloed clots. The animals were killed at the end of 4 h propofol infusion and lung specimens were obtained for determination of lung cell apoptosis rate by flow eytometry and expression of caspase-3, Bax, Bcl-2, Fas, FasL mRNA and protein by RT-PCR and Western blot.The expression of Bcl-2/Bax mRNA and protein was calculated. Results Compared with group S,APTE significantly increased the lung cell apoptosis rate, and expression of caspase-3, Bax, Fas, FasL and decreased the expression of Bcl-2 and Bcl-2/Bax. Propofol infusion significantly attenuated these APTE-induced changes. Conclusion Propofol can inhibit APTE-induced lung cell apoptosis by down-regulating the caspase-3, Fas and FasL expression and regulating the balance between Bcl-2 and Bax expression.     

异丙酚对氯胺酮致老龄大鼠认知功能障碍的影响

目的 探讨异丙酚对氯胺酮致老龄大鼠认知功能障碍的影响.方法 健康雄性SD老龄大鼠32只,月龄18～24月龄,体重380～470 g,随机分为4组(n=8):对照组(C组)、异丙酚组(P组)、氯胺酮组(K组)和异丙酚+氯胺酮组(PK组).P组静脉输注异丙酚30 mg·kg-1·h-1,K组静脉输注氯胺酮40 mg·kg-1·b-1,PK组静脉输注异丙酚30 mg·kg-1·h-1+氯胺酮40 mg·kg-1·h-1,C组给予等容量生理盐水,每天2 h,连续7 d.给药结束后测定大鼠认知功能,记录逃避潜伏期和穿越平台次数.认知功能测试完毕后,处死大鼠,取海马组织,检测CA1区神经元凋亡情况,计算神经元凋亡率;测定CA1区caspase-3的表达水平.结果 与C组比较,P组逃避潜伏期、穿越平台次数、海马CA1区神经元凋亡率和caspase-3表达差异无统计学意义(P＞0.05),K组和PK组逃避潜伏期延长,穿越平台次数减少,海马CA1区神经元凋亡率升高,caspase-3表达上调(P＜0.05);与K组比较,PK组逃避潜伏期缩短,穿越平台次数增加,海马CA1区神经元凋亡率降低,caspase-3表达下调(P＜0.05).结论 异丙酚可减轻氯胺酮致老龄大鼠认知功能障碍,其机制可能与异丙酚可在一定程度上抑制氯胺酮所致caspase-3表达上调,从而抑制氯胺酮诱发的神经元凋亡有关.     

凋亡相关蛋白及粘附分子在大鼠心脏急性排斥反应中的作用

目的 探讨凋亡及凋亡相关因子Fas／FasL、Bcl-2/Bax，粘附分子ICAM-1、P-selectin、E-selectin、L-Selectin、PECAM-1和VCAM-1在心脏移植中的表达及意义。方法 用免疫组织化学方法和原位杂交法，检测大鼠心脏移植后不同时间(1、3、5、7d)、Fas／FasL、ICAM-1、P-selectin、L-selectin、E-selectin蛋白、Bcl-2/Bax、VCAM-1、PECAM-1 mRNA的表达情况。结果 随着移植后天数的增加，细胞凋亡增多，Fas／FasL、Bax表达增加，粘附分子ICAM-1、VCAM-1、PECAM-1表达也增加，Selectin表达较少。正常大鼠肝脏未见细胞凋亡及粘附分子的表达。结论 细胞凋亡和粘附分子ICAM-1、VCAM-1、PECAM-1表达增加可能与心脏移植后急性排斥反应有关，而Fas／FasL、Bax可能促进了凋亡的发生。     

垂体中叶素对肾脏缺血再灌注大鼠肾小管上皮细胞凋亡的影响及其机制

目的 探讨垂体中叶素（IMD）对肾脏缺血再灌注（I/R）大鼠肾小管上皮细胞凋亡的影响及相关机制。 方法 健康雄性Wistar大鼠24只随机分为假手术组、I/R组、空质粒组、IMD质粒组。动物右肾切除后,采用超声微泡法,将空质粒或IMD质粒转染入左肾,1周后制作肾脏I/R模型。TUNEL法测定细胞凋亡;半定量RT-PCR检测Bax、Bcl-2、Fas的mRNA表达水平;比色法检测caspase-8、-9的活性;Western印迹法检测caspase-3蛋白的表达水平。 结果 与假手术组相比,I/R组细胞凋亡率增高,Bax、Fas mRNA表达增加,bcl-2 mRNA表达下降,caspase-8、-9活性增强,caspase-3蛋白表达增加（均P < 0.05）。与I/R组相比,IMD转染组细胞凋亡率明显降低,Bax、Fas的mRNA表达下降,bcl-2的mRNA表达增加,caspase-8、-9活性减弱,caspase-3蛋白表达减少（均P < 0.05）。转空质粒组与I/R组比较,各指标差异均无统计学意义。 结论 IMD能上调bcl-2表达,降低bax、Fas的表达,降低caspase-8、-9活性,从而抑制肾脏I/R损伤所诱导的凋亡。     

异丙酚预先给药对大鼠脑缺血再灌注时内质网应激性细胞凋亡的影响

目的 评价异丙酚预先给药对大鼠局灶性脑缺血再灌注损伤时内质网应激性细胞凋亡的影响.方法 健康成年雄性SD大鼠80只,体重240～280 g,采用随机数字表法,将大鼠随机分为4组(n=20):假手术组(S组);局灶性脑缺血再灌注组(FCIR组)采用阻塞大脑中动脉4h恢复灌注的方法制备大鼠局灶性脑缺血再灌注模型;异丙酚预先给药组(P组)于缺血前30 min股静脉泵注异丙酚12mg·kg-1·h-1至缺血15min;脂肪乳预先给药组(Ⅰ组)给予脂肪乳1.2 ml·kg-1·h-1.于再灌注6h时各组随机取10只大鼠采用Longa评分法行神经功能缺陷评分(NDS),处死取左侧大脑,用干湿重法测定脑含水量,余10只大鼠处死后取左侧海马,采用Western blot法检测C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白(CHOP)、Bcl-2和活化型caspase-3蛋白的表达水平.结果 与S组比较,FCIR组再灌注6h时NDS评分和脑含水量升高,CHOP和活化型caspase-3蛋白表达上调,Bcl-2蛋白表达下调,P组NDS评分升高(P＜0.05);与FCIR组比较,P组再灌注6h时NDS评分和脑含水量降低,CHOP和活化型caspase-3蛋白表达下调,Bcl-2蛋白表达上调(P＜0.05),Ⅰ组各指标差异无统计学意义(P＞0.05).结论 异丙酚可通过抑制内质网应激介导的细胞凋亡减轻大鼠局灶性脑缺血再灌注损伤.     

异丙酚对大鼠心肌缺血再灌注时Pim-1表达的影响

目的 探讨异丙酚对大鼠心肌缺血再灌注时Pim-1表达的影响.方法 健康雄性SD大鼠40只,体重220～250 g,采用随机数字表法,将其随机分为5组(n=8):假手术组(S组)、缺血再灌注组(I/R组)、脂肪乳剂组(I组)、低剂量异丙酚组(P1组)和高剂量异丙酚组(P2组).I/R组、I组、P1组和P2组和采用结扎左冠状动脉前降支30 min再灌注的方法制备心肌缺血再灌注损伤模型.P1组和P2组于缺血前10 min经股静脉输注异丙酚6、12 mg·kg-1·h-1至再灌注120 min,I组给予脂肪乳剂1.2 ml·kg-1 ·h-1.再灌注120 min时,取心肌组织,测定心肌梗死面积、细胞凋亡、Pim-1表达和caspase-3活性.结果 与S组比较,I/R组和I组心肌梗死面积、凋亡指数和caspase-3活性升高,心肌组织Pim-1表达下调(P＜0.01);与I/R组比较,P1组和P2组心肌梗死面积、凋亡指数和caspase-3活性降低,心肌组织Pim-1表达上调(P＜0.01),I组上述指标差异无统计学意义(P＞0.05).结论 异丙酚可减轻大鼠心肌缺血再灌注损伤,其机制与上调Pim-1表达有关.     

异丙酚对大鼠脑缺血再灌注后脑细胞凋亡和Bcl-2、Caspase-3蛋白表达的影响

脑缺血再灌注后脑损伤的发病机制较复杂,其所致病理损伤包括细胞坏死和凋亡。细胞凋亡与脑缺血再灌注损伤关系密切,是神经元迟发性死亡的主要形式。研究提示与凋亡密切相关的Bcl-2、Caspase-3蛋白等参与了这种迟发性死亡,它们表达的量决定了细胞的生存。本实验采用流式细胞仪、     

Emodin induces apoptosis in human prostate cancer cell LNCaP

AIM: To elucidate effects and mechanisms of emodin in prostate cancer cells. METHODS: Viability of emodin-treated LNCaP cells and PC-3 cells was measured by MTT assay. Following emodin treatments, DNA fragmentation was assayed by agarose gel electrophoresis. Apoptosis rate and the expression of Fas and FasL were assayed by flow cytometric analysis. The mRNA expression levels of androgen receptor (AR), prostate-specific antigen (PSA), p53, p21, Bcl-2, Bax, caspase-3, -8, -9 and Fas were detected by RT-PCR, and the protein expression levels of AR, p53 and p21 were detected by Western blot analysis. RESULTS: In contrast to PC-3, emodin caused a marked increase in apoptosis and a decrease in cell proliferation in LNCaP cells. The expression of AR and PSA was decreased and the expression of p53 and p21 was increased as the emodin concentrations were increased. In the same time, emodin induced apoptosis of LNCaP cells through the upregulation of caspase-3 and -9, as well as the increase of Bax /Bcl-2 ratio. However, it did not involve modulation of Fas or caspase-8 protein expression. CONCLUSION: In prostate cancer cell line, LNCaP, emodin inhibites the proliferation by AR and p53-p21 pathways, and induces apoptosis via the mitochondrial pathway.     

Apoptosis of T lymphocytes invading glioblastomas multiforme: a possible tumor defense mechanism

OBJECT: The goal of this study was to investigate whether apoptosis occurs in T lymphocytes that invade Fas ligand (FasL)-expressing glioblastomas multiforme (GBMs) and if its induction could be mediated by Fas. METHODS: Apoptotic T lymphocytes were detected in GBMs by using detection of cell-type markers combined with active caspase-3 immunohistochemical analysis, a recently introduced apoptosis-specific in situ ligation assay, as well as by examining morphological criteria. Apoptotic T cells expressed Fas and were localized in the vicinity or in direct contact with FasL-expressing tumor cells. The T lymphocytes were undergoing apoptosis in spite of Bcl-2 expression. Expression of Bax was also detected in dying T cells, which can explain the absence of the protective effect of Bcl-2. because Bax inhibits Bcl-2 death-repressor activity. CONCLUSIONS: On the basis of the data presented in this paper, the authors suggest that GBM cells that express FasL can induce apoptosis in invading immune cells. This phenomenon may play an important role in these tumors' maintenance of immune privilege and evasion of immune attacks. Awareness of this phenomenon should be helpful for the development of novel strategies for treatment of malignant gliomas.     

肾上腺髓质素对大鼠肾缺血再灌注损伤中肾小管上皮细胞凋亡的影响及机制研究

目的 观察肾上腺髓质素(adrenomedullin,AM)对大鼠肾缺血再灌注损伤(ischemia reperfusion injury,IRI)中肾小管上皮细胞凋亡的影响,并探讨其机制.方法 Wistar大鼠32只,随机分为4组:假手术组和IRI组各6只,转染空质粒组和转染AM质粒组各10只.大鼠右肾切除后1周,用超声微泡造影剂介导的基因转染方法将大鼠AM真核表达质粒转染大鼠肾脏,1周后采用免疫组织化学方法检测转染效率.转染成功后夹闭左肾动脉45 min制作肾IRI模型,于再灌注24 h后留取肾组织标本.TUNEL染色检测肾组织细胞凋亡,RT-PCR检测肾组织Bcl-2、Bax和Fas的mRNA表达,蛋白质印迹法检测caspase-3、caspase-8和caspase-9蛋白质表达.结果 转染AM质粒组的AM表达显著高于转染空质粒组(0.51±0.09和0.23±0.05,P＜0.05).与假手术组相比,IRI组肾组织细胞凋亡率明显增加[(38.79±7.52)%和(2.89±0.52)%,P＜0.05];肾组织Bax、Bcl-2、Fas、caspase-3、caspase-8和caspase-9表达上调,分别为0.72±0.18和0.23±0.04、0.80±0.12和0.38±0.06、1.24±0.25和0.39±0.09、0.76±0.13和0.38±0.08、0.92±0.14和0.32±0.06、0.89±0.12和0.42±0.09(P＜0.05),Bax/Bcl-2升高(0.91±0.18和0.61±0.08,P＜0.05).转染AM质粒组肾组织凋亡细胞数、Bax、Fas、caspase-3、caspase-8和caspase-9表达下调,分别为(19.36±6.78)%、0.48±0.11、0.62±0.07、0.53±0.08、0.46±0.08、0.51±0.12,与IRI组比较差异均有统计学意义(P＜0.05);Bcl-2表达进一步上调为1.23±0.25,Bax/Bcl-2降低为0.44±0.12,与IRI组比较差异均有统计学意义(P＜0.05).转染空质粒组和IRI组比较,上述各指标差异均无统计学意义(P＞0.05).结论 AM能减轻肾IRI引起的肾小管上皮细胞凋亡,其部分机制可能是通过抑制caspase依赖的内、外源性凋亡途径实现的.

Abstract：

Objective To investigate the effect and mechanism of adrenomedullin (AM) on apoptosis of renal tubular epithelial cell in rats induced by renal ischemia reperfusion injury. Methods Thirty-two Wistar rats were randomly divided into 4 groups: control group, IRI group, empty plasmid group and AM group. One week after removing the right kidney, eukaryotic expression vector encoding rat AM gene was transfected into the left kidney using an ultrasound-microbubble mediated system. After 1 week the transfer efficiency was detected by immunohistochemical method . Renal IRI model induced by clamping left renal arteries for 45 min followed by reperfusion for 24 h. Tubular cell apoptosis was detected by TUNEL assay. Bcl-2, Bax and Fas expressions were examined by RT-PCR. The expressions of caspase-3, caspase-8 and caspase-9 were determined by Western bolt analysis. Results The expression of AM in the AM group was significantly higher than the empty plasmid group (0.51±0.09 vs 0.23±0.05; P<0.05). Compared with the control group, the apoptosis rate of renal tubular cell in the IRI group was significantly higher [(38.79±7.52)% vs (2.89±0.52)%; P<0.05]. The expressions of Bax, Bcl-2, Fas, caspase-3, caspase-8 and caspase-9 were also significantly increased (0.72±0.18 vs 0.23±0.04, 0.80±0.12 vs 0.38±0.06, 1.24±0.25 vs 0.39±0.09, 0.76±0.13 vs 0.38±0.08, 0.92±0.14 vs 0.32±0.06, 0.89±0.12 vs 0.42±0.09; P<0.05). Bax/Bcl-2 was also significantly increased (0.91±0.18 vs 0.61±0.08; P<0.05). Compared with the IRI group, AM pretreatment significantly decreased the apoptosis rate of renal tubular cells [(19.36±6.78)% vs (38.79±7.52)%; P<0.05]. AM inhibited the up-regulation of Bax, Fas, caspase-3, caspase-8 and caspase-9, while promoting the up-regulation of Bcl-2 (0.48±0.11 vs 0.72±0.18, 0.62±0.07 vs 1.24±0.25, 0.53±0.08 vs 0.76±0.13, 0.46±0.08 vs 0.92±0.14, 0.51±0.12 vs 0.89±0.12, 1.23±0.25 vs 0.80±0.12; P<0.05). Bax/Bcl-2 significantly decreased (0.44±0.12 vs 0.91±0.18; P<0.05). The above parameters had no significant diffe-rence between the empty plasmid group and the IRI group (P>0.05). Conclusion AM can reduce apoptosis of renal tubular epithelial cell induced by renal IRI, the mechanism of which might be achieved by inhibiting caspase-dependent intrinsic and extrinsic pathways.     

反义K-ras基因对胰腺癌细胞增殖和凋亡的影响

目的 探讨反义K-ras癌基因对胰腺癌细胞增殖和凋亡及Fas/FasL、Bcl-2、Bax表达的影响.方法 将重组逆转录病毒载体pLXSN转染包装细胞PT-67,获得重组逆转录病毒.在细胞和动物水平将该重组逆转录病毒分别感染PC-3和BxPC-3胰腺癌细胞,经G418筛选获得稳定细胞系,应用MTT、流式细胞仪和免疫组化法分别研究其增殖、凋亡及其相关基因表达情况.结果 成功制备含反义K-ras基因的重组逆转录病毒.感染含反义K-ras基因重组病毒后,胰腺癌PC-3细胞和移植瘤(有K-ras基因第12密码子点突变GGT→GTT)增殖受到抑制,G<,0/1>期细胞增加而S期细胞明显减少,细胞凋亡增加.免疫组化显示含反义K-ras癌基因逆转录病毒感染后PC-3细胞Bax/Bcl-2比值升高,Fas表达上调.结论 反义K-ras基因可使胰腺癌细胞及移植瘤增殖受到抑制,并可通过上调Bax/Bcl一2比值和(或)促进Fas表达诱导细胞凋亡.     

Intracellular mechanisms of cyclosporin A-induced tubular cell apoptosis

Tubular cell apoptosis contributes to the pathogenesis of renal injury. However, the intracellular pathways that are active in tubular epithelium are poorly understood. The lethal pathways activated by cyclosporin A (CsA), a nephrotoxin that induces caspase-dependent apoptosis in tubular epithelium, were explored. Fas expression, caspase activation, and mitochondrial injury were assessed by Western blot, flow cytometry, and microscopy in cultured murine tubular epithelial cells exposed to CsA. The influence of FasL antagonists, Bax antisense oligodeoxynucleotides, and caspase inhibitors on cell survival was explored. Tubular cells constitutively express FasL. CsA increased the expression of Fas. However, Fas had no role in CsA-induced apoptosis, as CsA did not sensitize to FasL-induced apoptosis, caspase-8 activity was not increased, and neither blocking anti-FasL antibodies nor caspase-8 inhibition prevented CsA-induced apoptosis. Apoptosis induced by CsA is associated with the translocation of Bax to the mitochondria and Bax antisense oligodeoxynucleotides protected from CsA-induced apoptosis. CsA promoted a caspase-independent release of cytochrome c and Smac/Diablo from mitochondria. CsA also led to a caspase-dependent loss of mitochondrial membrane potential. Caspase-2, caspase-3, and caspase-9 were activated, and specific caspase inhibitor prevented apoptosis and increased long-term survival. Evidence for endoplasmic reticulum stress, such as induction of GADD153, was also uncovered. However, endoplasmic reticulum-specific caspase-12 was not activated. CsA induces changes in several apoptotic pathways. However, the main lethal apoptotic pathway in CsA-exposed tubular epithelial cells involves mitochondrial injury.     

自然流产模型小鼠绒毛滋养层细胞凋亡及相关调控蛋白的表达

目的探讨自然流产模型小鼠绒毛滋养层细胞凋亡异常与自然流产的关系。方法建立正常妊娠模型CBAXBALB/c和自然流产模型CBAXDBA/2。采用DNA缺口原位末端标记技术(TUNEL)测定两组模型孕13 d绒毛滋养层细胞凋亡情况;免疫组织化学SABC法测定两组模型孕13 d绒毛组织Bcl-2、Bax、Fas、FasL 4种凋亡调控蛋白的表达,并以MBIS-2000医用彩色病理图像免疫组织化学测量系统对其表达进行半定量分析,结果用平均灰度值表示。结果自然流产模型小鼠绒毛滋养层细胞凋亡指数明显高于正常妊娠模型(P<0.01)。Bax表达亦高于正常妊娠模型(P<0.05);FasL表达低于正常妊娠模型(P<0.01);Fas和Bcl-2表达两组无明显差异。结论早孕期绒毛滋养层细胞凋亡异常是自然流产机制之一,Bcl-2/Bax,Fas/FasL途径可能是诱导早孕期绒毛滋养层细胞凋亡的重要因素。     

他莫昔芬与γ-干扰素联合抗人乳腺癌细胞株的作用及其机制研究

目的: 探讨他莫昔芬(TAM)与 γ-干扰素(γ-IFN)联合抗乳腺癌细胞株的作用及其机制。方法:在体外培养条件下，分别或联合应用γ-IFN,TAM或雌二醇（E2）作用于ER阳性的MCF-7人乳腺癌细胞株，用MTT比色法分析细胞生长抑制作用;用流式细胞仪（FCM）检测细胞周期分布、凋亡率及用药前后Bcl-2,Bax,Fas,FasL及Caspase-8蛋白的变化;荧光分光光度仪检测Caspase-3活性。结果:TAM能抑制ER阳性乳腺癌细胞的生长，阻滞细胞周期于G0/G1期，并可诱导细胞凋亡;同时，TAM能拮抗外源性雌激素对MCF-7细胞的促生长作用。γ-IFN预处理细胞24h后，TAM抗乳腺癌细胞的作用增强。联用γ-IFN与TAM后，细胞Bcl-2蛋白表达下调，Caspase-8表达上调;但药物处理前后，细胞Bax，Fas，FasL蛋白表达水平及Caspase-3活性未见明显变化。结论:体外条件下，TAM通过影响细胞周期、诱导细胞凋亡而发挥抗ER阳性乳腺癌细胞作用;γ-IFN能加强TAM的抗乳腺癌作用。两者作用机制可能系通过下调Bcl-2表达和上调Caspase-8的表达。     

Role of E-selectin in cell apoptosis induced by allogeneic blood perfusion in isolated mouse lung

BACKGROUND: In a model of mouse isolated lung, we have recently demonstrated that E-selectin is involved in the activation of endothelial cells induced by allogeneic blood perfusion. In the present study, we explored the signaling pathway of apoptosis induced by E-selectin triggering. METHODS: Lungs were perfused for 3 hours with fresh blood in the absence or presence of an anti-E-selectin monoclonal antibody, or a protein kinase C (PKC), protein tyrosine phosphatase (PTP), or protein tyrosine kinase (PTK) inhibitor. The number of apoptotic cells in lung sections was determined by a TUNEL method. mRNAs for Fas, FasL and caspase-8, and for Bad, Bax, Bcl-w, Bcl-xL and caspase-9, for the FasL and the mitochondrial cytochrome-c pathways of apoptosis, respectively, and mRNA for the effector caspase-3 were quantified in lung tissues by RT-PCR. PTP and Src-PTK activities were also measured. RESULTS: After 3 hours of allogeneic perfusion, we observed a significant increase in: 1) the number of apoptotic cells in lung sections, 2) mRNA levels of FasL, Bcl-xL, caspase-8 and caspase-3, and 3) PTP activity (P < 0.05 compared with isogeneic perfusion). Surprisingly, mRNA levels of the proapoptotic genes Bad and Bax were significantly decreased (P < 0.05). PTK activity and caspase-9 mRNA level were not affected. Blocking anti-E-selectin mAbs and inhibitors for PKC, PTP, and PTK resulted in a significant reduction of apoptosis. CONCLUSIONS: In our model, the engagement of E-selectin induced by endothelial cell allogeneic activation appeared to be a prerequisite for lung apoptosis, which involved FasL and increase of PTP activity. Blockade of apoptosis with selective inhibitors may be a promising approach to the treatment/prevention of lung graft injury.